Project Summary/Abstract Activating mutations in KRAS, BRAF and EGFR are the underling cause of a large number of deadly human cancers. A single missense amino acid mutation in these genes causes cancers of diverse origins including a large number of pancreatic, lung, skin, and colorectal cancers. The general signaling pathway affected by these mutations, the so-called RAS-RAF-MEK-ERK pathway (ERK signaling), is a major target of drug design by pharmaceutical companies. However, regardless of the molecular target (activated BRAF or EGFR, for example), this approach has been hindered due to the emergence of drug resistance in nearly all cases. To tackle this important human disease there is a need for new groundbreaking unconventional approach to cancer treatment. We propose to establish a potentially transformative approach to treating these cancers, which we refer to as Enhancer RNA Therapy. Indeed, we believe this plan will be applicable to a wide spectrum of human diseases. Our proposal hinges on our discovery of the functional importance of enhancer RNAs (eRNAs); noncoding RNAs that are transcribed from tissue and disease-specific enhancers. Enhancers are DNA elements that govern spatial and temporal regulation of gene expression during development and disease. Aberrant regulation of enhancers is considered to be a key event in the genesis and progression of cancer. We aim to target disease-induced eRNAs to silence cancer-causing genes as a transformative treatment in cancers induced by activating mutations of KRAS, BRAF and EGFR. We will develop an atlas of disease-induced enhancers in multiple cancers with activating mutations in KRAS and BRAF. We will then functionally characterize these cancer-induced enhancers using genetic and functional approaches. Finally, in collaboration with Isis Pharmaceuticals, we will develop derivatized anti-sense oligonucleotides (ASOs) that will neutralize the oncogenic eRNAs in vivo allowing for tissue specific inhibition of pathogenic gene expression in cancer. We therefore propose to investigate the feasibility of 'Enhancer RNA Therapy' using cancers with activating mutations in ERK signaling pathway as a model system.